The long term goal of this research is to elucidate the molecular architecture of the mitochondrial outer membrane. During the project period of two years, we will focus our attention on the protein (enzyme) topography of the membrane. Using two powerful analytical tools, a two dimensional gel electrophoresis method to separate polypeptides and an ultrasensitive silver staining technique to detect proteins, we have resolved, according to isoelectric pH and molecular weight, the polypeptides of the membrane. We now intend to identify a few of the spots with protein samples we have obtained from other investigators. Completion of this groundwork will lead us to the central aspect of the problem, viz., protein topography of the outer membrane. We have standardized methods to probe the outer and also the inner surface of the outer membrane. The approaches to be taken are (a) action of different proteolytic enzymes, (b) lactoperoxidase iodination technique and, (c) antigen-antibody reaction mainly for glycerophosphate acyltransferase. At the end of two years, we expect to have identified, on the basis of isoelectric pH and molecular weight, which proteins are exposed on the outer surface, which are present in the inner side and which span the transverse plane of the outer membrane. In another approach, we will focus our attention on a few outer membrane enzymes. We intend to purify mitochondrial glycerophosphate acyltransferase and to examine closely its topography. Furthermore, we hope to make significant progress in localizing mitochondrial acyl-CoA ligase, monoacylglycerophosphate acyltransferase, kynurenine hydroxylase and rotenone-insensitive NADH-cytochrome c reductase, in the transverse plane of the outer membrane. It is expected that an elucidation of the molecular architecture of the outer membrane will help understand how this boundary structure of the mitochondria interacts with the inner membrane, cytosol or other cellular compartments.